Effect of the Secretory Small GTPase Rab27B on Breast Cancer Growth, Invasion, and Metastasis

Abstract

BACKGROUND Secretory GTPases like Rab27B control vesicle exocytosis and deliver critical proinvasive growth regulators into the tumor microenvironment. The expression and role of Rab27B in breast cancer were unknown. METHODS Expression of green fluorescent protein (GFP) fused with wild-type Rab3D, Rab27A, or Rab27B, or Rab27B point mutants defective in GTP/GDP binding or geranylgeranylation, or transient silencing RNA to the same proteins was used to study Rab27B in estrogen receptor (ER)-positive human breast cancer cell lines (MCF-7, T47D, and ZR75.1). Cell cycle progression was evaluated by flow cytometry, western blotting, and measurement of cell proliferation rates, and invasion was assessed using Matrigel and native type I collagen substrates. Orthotopic tumor growth, local invasion, and metastasis were analyzed in mouse xenograft models. Mass spectrometry identified proinvasive growth regulators that were secreted in the presence of Rab27B. Rab27B protein levels were evaluated by immunohistochemistry in 59 clinical breast cancer specimens, and Rab3D, Rab27A, and Rab27B mRNA levels were analyzed by quantitative real-time polymerase chain reaction in 20 specimens. Statistical tests were two-sided. RESULTS Increased expression of Rab27B promoted G(1) to S phase cell cycle transition, proliferation and invasiveness of cells in culture, and invasive tumor growth and hemorrhagic ascites production in a xenograft mouse model (n = 10; at 10 weeks, survival of MCF-7 GFP- vs GFP-Rab27B-injected mice was 100% vs 62.5%, hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.88, P = .03). Mass spectrometric analysis of purified Rab27B-secretory vesicles identified heat-shock protein 90alpha as key proinvasive growth regulator. Heat-shock protein 90alpha secretion was Rab27B-dependent and was required for matrix metalloproteinase-2 activation. All Rab27B-mediated functional responses were GTP- and geranylgeranyl-dependent. Presence of endogenous Rab27B mRNA and protein, but not of Rab3D or Rab27A mRNA, was associated with lymph node metastasis (P < .001) and differentiation grade (P = .001) in ER-positive human breast tumors. CONCLUSIONS Rab27B regulates invasive growth and metastasis in ER-positive breast cancer cell lines, and increased expression is associated with poor prognosis in humans.

Multiple uses of basement membrane‐like matrix (BME/Matrigel) in vitro and in vivo with cancer cells

“…The activity of various inhibitory or stimulatory agents has been assessed in vitro in the invasion assay, 34 and in many cases the active reagents have efficacy in animal models of tumor growth and metastasis. 29,35 The assay can be used to identify genes (by transfection or silencing) that regulate invasion/malignancy and thus provide rationale for therapeutic strategies. 33,36 For example, heparanase has been proven to be important in promoting tumor growth and metastasis, 37 and silencing this enzyme reduces invasion through basement membrane extract, 36 thus, providing an in vitro model to evaluate therapeutics before expensive in vivo testing.…”

Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis

“…An increased expression of Rab27B is associated with the poor prognosis of oestrogen receptor-positive breast cancer patients. Through mass spectrometric (MS) analysis, heat-shock protein 90a has been identified as a component of Rab27B-regulated vesicles and it acts as a pro-invasive growth regulator required for MMP2 activation 6 . Previous studies indicate that Rab25 functions as a tumour suppressor or oncogene depending on the cancer type [10][11][12] .…”

On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

“…In a next step, on-chip fSPT size and concentration measurements were performed of cellderived MVs isolated from the conditioned cell culture medium of breast cancer cells 28 (see Supporting Video 2). An excess of fluorescently labelled Annexin V was used to label the cell-derived MVs which are known to expose phosphatidylserine (PS) on their surface 13 , followed by on-chip fSPT analysis without additional purification.…”